Laboratory studies on salmonella-contaminated cheese involved in a major outbreak of gastroenteritis

A major outbreak of gastroenteritis was traced to Cheddar cheese contaminated with Salmonella typhimurium. There were no significant differences in pH values of the contaminated (mean pH 5.31) and non-contaminated (mean pH 5.39) cheese. The isolation rates of Salm. typhimurium were about the same when cheese samples were homogenized in lactose broth, lactose broth containing 1% Tween 80, or in aqueous 2% sodium citrate. Salmonella typhimurium was isolated regardless of preenrichment in lactose broth, but required selective enrichment in selenite cystine or tetrathionate brilliant green broth. There were no marked differences in the isolation rates obtained with different selective enrichment media, or after incubation at 36 degrees and 43 degrees C for 24 or 48 h. Contaminated samples of cheese failed to yield Salm. typhimurium consistently despite large and multiple samplings; samples from the interior of cheese blocks yielded positive results more frequently than the samples from the exterior. The number of Salm. typhimurium in factory sealed blocks as well as in samples obtained from the homes of known cases of salmonellosis was found to range from less than 3/100 g to 9/100 g of cheese. The infective dose of Salm. typhimurium in contaminated cheese was probably no greater than 10(4) organisms, and a rapid decline in numbers of Salm. typhimurium must have occurred subsequent to the outbreak.     

Laboratory studies on salmonella-contaminated cheese involved in a major outbreak of gastroenteritis

Dried biomass of Streptomyces cyaneus NCIB 9616 harvested at 1, 3, 7, 10 and 14 d from a liquid culture was degraded by alkaline methanolysis and the fatty acid methyl esters released examined quantitatively by gas chromatography. Isoprenoid quinones were extracted with petroleum ether and methanolic saline, purified by thin-layer chromatography and quantified by high performance liquid chromatography. Data for each component were plotted against time. The results showed that at the beginning of logarithmic growth there was a small quantitative shift in the fatty acid composition but, the fatty acid profiles remained relatively constant during both the logarithmic and stationary phases of growth. In contrast, the isoprenoid quinone composition depended markedly on the stage of growth. These results indicate that valid comparison of isoprenoid quinone profiles requires that sampling take place at defined stages of the growth cycle.     

Electrostatic interactions in sperm whale myoglobin. Site specificity, roles in structural elements, and external electrostatic potential distributions

The electrostatic free energy contribution to the stability of sperm whale ferrimyoglobin was evaluated according to the static accessibility modified Tanford-Kirkwood model. The electrostatic free energy contribution of each distinct structural element was divided into one term arising from interactions between it and other elements (interelemental) and another from interactions within the particular element itself (intraelemental). At pH 7 the majority of the terms were found to be stabilizing. The interelemental terms are the dominant ones for most structural elements. The small interelemental terms of the C and D helices are compensated by large intraelemental interactions which stabilize these short helices. Perturbations in pH can be accommodated by the structural elements through a redistribution of stabilizing and destabilizing interactions. The electrostatic potentials calculated at the surface of the protein indicate that the internal compensation of local potentials achieved during folding results in a generally neutral protein-solvent interface save for two distinct areas of nonzero potential. The accessibility of each charged atom to solvent was analyzed in terms of the surface area lost to charged, polar and nonpolar atoms separately. The net solvent accessibility lost parallels closely that lost to nonpolar atoms alone, indicating a specific role for nonpolar atoms in defining dielectric shielding of charged atoms, aside from their participation in the well-known hydrophobic interactions.     

pH-dependent processes in proteins

Recent improvements in the understanding of electrostatic interactions in proteins serve as a focus for the general topic of pH-dependent processes in proteins. The general importance of pH-dependent processes is first set out in terms of hydrogen ion equilibria, stability, ligand interactions, assembly, dynamics, and events in related molecular systems. The development of various theoretical treatments includes various formalisms in addition to the solvent interface model developed by Shire et al. as an extension of the Tanford-Kirkwood treatment. A number of detailed applications of the model are presented and future potentialities are sketched.     

Bacterial Infection of Washed and Unwashed Eggs with Reference to Salmonellae

A survey of eggs in the Fraser Valley area of British Columbia was conducted to determine the level of bacterial contamination on washed and unwashed shell eggs and the incidence of Salmonella infection in shell eggs. Samples of eggs were taken from 15 grading stations. Determinations of eggs from 87 shipments showed that a higher proportion of eggs was heavily contaminated (>5-million organisms per egg) after washing than as received at the grading stations. This finding suggests the need for the development of more satisfactory washing procedures. No salmonellae were detected in the 180 samples (3,995 eggs) which were examined. It was concluded that Salmonella contamination of intact shell eggs does not constitute a serious health hazard in eggs from this area.     

A possible role for the pcnB gene product of Escherichia coli in modulating RNA: RNA interactions.

Summary The sequence of the PcnB protein of Escherichia coli, a protein required for copy number maintenance of ColE1-related plasmids, was compared with the PIR sequence database. Strong local similarities to the sequence of the E. coli protein tRNA nucleotidyltransferase were found. Since a substrate of the latter protein, tRNA, structurally resembles the RNAs that control ColE1 copy number we believe that we may have identified a region in PcnB that interacts with these RNAs. Consistent with this idea is our observation that PcnB is required for the replication of R1, a plasmid whose replication is also regulated by a small RNA.     

GTPase-dependent signaling in bacteria: characterization of a membrane-binding site for era in Escherichia coli.

Era is an Escherichia coli GTPase that is essential for cell viability and is peripherally associated with the cytoplasmic membrane. Both immunoelectron microscopy and subcellular-fractionation experiments have shown that Era is present in cytoplasmic as well as membrane-associated pools. These data led to speculation that the mechanism of action of Era may require cycling between membrane and cytoplasmic sites. In order to investigate this possibility, an in vitro binding assay was developed to characterize the binding of Era to membrane fractions. Competition and saturation binding experiments suggest that a site that is specific for Era and capable of binding up to 5 ng of Era per microgram of membrane protein is present in membrane preparations. The binding curve is complex, indicating that multiple equilibria describe the interaction. The binding of Era to this putative receptor is dependent on guanine nucleotides; binding cannot be measured in the absence of nucleotide, and neither ATP nor UTP can substitute. Subfractionation of cell walls showed that the guanine nucleotide-dependent binding site was present in fractions enriched in cytoplasmic membrane. These data provide evidence that Era may be involved in a GTPase-receptor-coupled membrane-signaling pathway that is essential for growth in E. coli.     

Substrate specificity of the protease that processes human interleukin-1 beta

The substrate specificity of the protease which generates mature human interleukin-1 beta (IL-1 beta) from pro-interleukin-1 beta was investigated using synthetic peptide substrates and recombinant pro-IL-1 beta. The requirement of an L-aspartate in the P-1 position was confirmed together with the need for a small hydrophobic residue in the P-1' position (Gly or Ala). It was shown that the enzyme can tolerate conservative substitutions in the P-2 and P-2' positions. We found little difference in the enzyme's ability to cleave denatured and native pro-IL-1 beta, indicating that tertiary structure recognition is not involved in binding. The enzyme did, however, require a peptide of more than six amino acids for cleavage to occur. These results conclusively demonstrate the unusual specificity of this protease.     

The purification and characterization of homologous high molecular weight storage proteins from grain of wheat,rye and barley

Summary Homologous high molecular weight storage prolamins were purified from grain of wheat, rye and barley using combinations of gel filtration, ion-exchange chromatography and preparative isoelectric focusing. Sodium dodecylsulphate polyacrylamide gel electrophoresis showed that the components were single bands with apparent mol.wts. of above 100,000. Molecular weights determined by sedimentation equilibrium ultracentrifugation were considerably lower; 54,700, 67,600 and 69,600 for the components from barley, rye and wheat respectively. Amino acid analysis showed the presence of 13.6 to 16.5 mol% glycine, 29.6 to 34.0 mol% glutamate + glutamine, 11.4 to 13.7 mol% proline and a total of 4.0 to 5.7 mol% basic amino acids. Automated N-terminal amino acid sequencing of the component from wheat showed the presence of cysteine residues at positions 5 and 10, and this is discussed in relation to the possible role of these proteins in the visco-elastic gluten network.